Our earlier work (Freedlender, Taichman and Smithies, Biochem. 16, 1802 (1977)) has shown that during subsequent cell divisions about 10% of the newly synthesized proteins associated with DNA in eukaryotic cells remain with the DNA strand synthesized in the same generation. We now propose an investigation of the nature of these proteins and their relationship to the maintenance of the differentiated state. Our earlier work was deliberately performed with protein rich chromatin using a BrUdRib ultraviolet light procedure. In the new work we will first confirm these results using more defined starting materials, monomeric and oligomeric subunits of chromatin, and a separation by density. Conditions for separating reversibly cross-linked material in density gradients of Cs salts will be developed using DNA labeled chromatin. Next mixing experiments will test for the possibility of exchange of proteins. Then cells will be labeled with radioactive amino acids and 5-bromodeoxyuridine (BrUdRib) so that any segregating proteins will be 14C labeled when associated with the grandparental strand of DNA and 3H labeled when associated with the parental strand. The distribution of labeled proteins to the daughter chromatin will the be followed to test for segregation. The pattern obtained with monomomers which contain only the four core histones will be compared with that obtained will oligomeric and polymeric nucleosomes. SDS and acid urea gel electrophoresis will be used in conjunction with these three preparations labeled with lysine or tryptophon (a nonhistone specific label) to answer the following questions: 1) Do any of the histones segregate? 2) If so, does a fraction of all the histones segregate? or 3) Do only specific histones segregate? 4) Do any non-histones segregate? The segregation behavior of adenovirus and SV40 proteins which bind tightly near the origin of DNA replication will be examined and compared to that of tight binding CHO proteins. If non-histones are segregating, we will try to isolate and characterize them.